Isolated bis-linezolid, preparation thereof, and its use as a reference standard

ABSTRACT

The present invention provides an isolated linezolid (1) impurity, bis-linezolid (4), preparation thereof and its use as a reference standard.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of provisional applications Ser.Nos. 60/656,778, filed Feb. 24, 2005, 60/656,646, filed Feb. 24, 2005,as well as 60/690,822, filed Jun. 14, 2005 which are incorporated hereinby reference.

FIELD OF THE INVENTION

The present invention relates to isolated bis-linezolid, methods ofpreparing and detecting bis-linezolid, and methods of usingbis-linezolid as a reference standard.

BACKGROUND OF THE INVENTION

Linezolid[(S)-N-[[3-(3-Fluoro-4-morpholinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]acetamide]is an antimicrobial agent. Linezolid is an oxazolidinone, having theempirical formula C₁₆H₂₀FN₃O₄ and the following structure (1):

Linezolid (1) is described in The Merck Index (13th edition, Monographnumber: 05526, CAS Registry Number: 165800-03-3) as white crystals, witha melting point of 181.5-182.5°. Linezolid (1), as well as a process forits preparation, is disclosed in U.S. Pat. No. 5,688,792 (Example 5),European Patent No. 717738, Israeli Patent No. 110,802, Canadian PatentNo. 2,168,560, and International Patent Publication WO 95/07271.

Linezolid (1) is marketed in the United States by Pfizer, Inc. as aninjection, tablets, and oral suspension under the name ZYVOX®. Its mainindications are nosocomial pneumonia, skin and skin-structureinfections, and vancomycin-resistant Enterococcus faecium infections.

U.S. Pat. No. 5,688,792 claims linezolid (1) and its use for thetreatment of microbial infections. This patent also discloses, but doesnot claim, the following method of preparation:

This method of preparation was also disclosed in Bricker, et al., J.Med. Chem., 39 673 -679 (1996), where it was stated that the above routeavoids the use of phosgene to make the carbamate precursor of theoxazolidinone ring. The authors also disclose that the use of NaN₃ canbe avoided by using potassium phthalimide, followed by deblocking of thephthalimide with aqueous methyl amine.

In the above-described synthesis, the intermediate amine,S-N-(4-morpholinyl-3-fluorophenyl)-2-oxo-5-oxazolidinyl-methyl amine (2)

is reacted without isolation with acetic anhydride as an oily product,or in solution, to produce the acetamide, linezolid (1). This isfollowed by procedures for isolating the linezolid (1) such as thosedescribed in U.S. Pat. No. 5,688,792, at col. 15, 11. 22-28(chromatography and separation of the desired fraction, followed byevaporation and trituration of the product to obtain pure linezolid(1)).

In the above-described syntheses, the intermediate azideR-N-(4-morpholinyl-3-fluorophenyl)-2-oxo-5-oxazolidinyl-methyl azide (3)

is reduced to its corresponding amine,S-N-(4-morpholinyl-3-fluorophenyl)-2-oxo-5-oxazolidinyl-methyl amine (2)in the solvent ethyl acetate by hydrogenation using hydrogen gas and apalladium/carbon catalyst. These reaction conditions lead to theproduction of an undesirable level of reaction by-products, and,following the acetylation of the intermediate amine (2) to linezolid(1), to undesirably high levels of bis-linezolid (4)

It is well known in the art that, for human administration, safetyconsiderations require the establishment, by national and internationalregulatory authorities, of very low limits for identified, buttoxicologically uncharacterized impurities, before an activepharmaceutical ingredient (API) product is commercialized. Typically,these limits are less than about 0.15 percent by weight of eachimpurity. Limits for unidentified and/or uncharacterized impurities areobviously lower, typically, less than 0.1 percent by weight. Therefore,in the manufacture of APIs, the purity of the products, such aslinezolid (1), is required before commercialization, as is the purity ofthe active agent in the manufacture of formulated pharmaceuticals.

It is also known in the art that impurities in an API may arise fromdegradation of the API itself, which is related to the stability of thepure API during storage, and from the manufacturing process, includingthe chemical synthesis. Process impurities include unreacted startingmaterials, chemical derivatives of impurities contained in startingmaterials, synthetic by-products, and degradation products.

In addition to stability, which is a factor in the shelf life of theAPI, the purity of the API produced in the commercial manufacturingprocess is clearly a necessary condition for commercialization.Impurities introduced during commercial manufacturing processes must belimited to very small amounts, and are preferably substantially absent.For example, the ICH Q7A guidance for API manufacturers requires thatprocess impurities be maintained below set limits by specifying thequality of raw materials, controlling process parameters, such astemperature, pressure, time, and stoichiometric ratios, and includingpurification steps, such as crystallization, distillation, andliquid-liquid extraction, in the manufacturing process.

The product mixture of a reaction is rarely a single compound withsufficient purity to comply with pharmaceutical standards. Side productsand by-products of the reaction and adjunct reagents used in thereaction will, in most cases, also be present in the product mixture. Atcertain stages during processing of an API, such as linezolid (1), theAPI must be analyzed for purity, typically, by HPLC or GC analysis, todetermine if it is suitable for continued processing and, ultimately,for use in a pharmaceutical product. The API need not be absolutelypure, as absolute purity is a theoretical ideal that is typicallyunattainable. Rather, purity standards are set with the intention ofensuring that an API is as free of impurities as possible, and, thus, isas safe as possible for clinical use. As discussed above, in the UnitedStates, the Food and Drug Administration guidelines recommend that theamounts of some impurities be limited to less than 0.1 percent.

Generally, side products, by-products, and adjunct reagents(collectively “impurities”) are identified spectroscopically and/or withanother physical method, and then associated with a peak position, suchas that in a chromatogram, or a spot on a TLC plate. (Strobel, H. A.;Heineman, W. R., Chemical Instrumentation: A Systematic Approach, 3rddd. (Wiley & Sons: New York 1989) (hereinafter “Strobel”), page 953).Thereafter, the impurity can be identified, e.g., by its relativeposition in the chromatogram, where the position in a chromatogram isconventionally measured in minutes between injection of the sample onthe column and elution of the particular component through the detector.The relative position in the chromatogram is known as the “retentiontime.” The retention time varies daily, or even over the course of aday, based upon the condition of the instrumentation, as well as manyother factors. To mitigate the effects such variations have uponaccurate identification of an impurity, practitioners use the “relativeretention time” (“RRT”) to identify impurities. (Strobel, page 922). TheRRT of an impurity is its retention time divided by the retention timeof a reference marker. In theory, linezolid (1) itself could be used asthe reference marker, but as a practical matter it is present in such alarge proportion in the mixture that it can saturate the column, leadingto irreproducible retention times, as the maximum of the peak can wander(Strobel, FIG. 24.8(b), page 879, illustrates an asymmetric peakobserved when a column is overloaded). Thus, it may be advantageous toselect a compound other than the API that is added to, or present in,the mixture in an amount sufficiently large to be detectable andsufficiently low as not to saturate the column, and to use that compoundas the reference marker.

Those skilled in the art of drug manufacturing research and developmentunderstand that a compound in a relatively pure state can be used as a“reference standard.” A reference standard is similar to a referencemarker, which is used for qualitative analysis only, but is used toquantify the amount of the compound of the reference standard in anunknown mixture as well. A reference standard is an “external standard”when a solution of a known concentration of the reference standard andan unknown mixture are analyzed using the same technique. (Strobel page924; Snyder, L. R.; Kirkland, J. J. Introduction to Modern LiquidChromatography, 2nd ed. (John Wiley & Sons: New York 1979) (hereinafter“Snyder”), page 549). The amount of the compound in the mixture can bedetermined by comparing the magnitude of the detector response to thereference standard and to the compound in the mixture. See also U.S.Pat. No. 6,333,198, incorporated herein by reference.

The reference standard can also be used to quantify the amount ofanother compound in the mixture if a “response factor,” whichcompensates for differences in the sensitivity of the detector to thetwo compounds, has been predetermined. (Strobel page 894). For thispurpose, the reference standard is added directly to the mixture, and isknown as an “internal standard.” (Strobel page 925, Snyder page 552).

The reference standard can even be used as an internal standard when,without the addition of the reference standard, an unknown mixturecontains a detectable amount of the reference standard compound using atechnique known as “standard addition.” In a “standard addition,” atleast two samples are prepared by adding known and differing amounts ofthe internal standard. (Strobel pp. 391-393, Snyder pp. 571, 572). Theproportion of the detector response due to the reference standardpresent in the mixture without the addition can be determined byplotting the detector response against the amount of the referencestandard added to each of the samples, and extrapolating the plot tozero. (See, e.g., Strobel, FIG. 11.4 page 392).

There is a need to isolate the bis-linezolid (4) impurity. This impuritymay also be used as a reference marker and/or standard.

SUMMARY OF THE INVENTION

In one embodiment, the invention is directed to isolated bis-linezolid(4), characterized by data selected from: a ¹H NMR spectrum (400 MHz,D₂O+TFA) δ (ppm):2.08 (s), 3.63 (m), 3.83 (t), 4.02 (s), 4.14 (m), 4.94(m), 7.23 (d), 7.58 (m); a ¹³C NMR spectrum (400MHz, CDCl₃) δ (ppm):16.6(q), 43-50 (s), 50.4 (s), 50.9 (s), 68.3 (s), 103.6, 104.0 (d), 111.1(s), 118.7 (s), 130-140 (s), 149-153 (d), 171.4 (s); FAB+m/z (MH⁺): 616;and an IR spectrum at 1519, 1572, 1644, 1743, 2825, 2858, 2891, 2965cm⁻¹.

In another embodiment, the invention is directed to the preparation ofbis-linezolid (4) by a method comprising converting the azideintermediate to linezolid (1) enriched in bis-linezolid (4), andisolating the bis-linezolid (4) impurity.

In yet another embodiment, the invention is directed to a method ofusing bis-linezolid (4) as a reference standard to analytically quantifythe purity of linezolid (1), and to set specific limits to the amount ofbis-linezolid (4) and other impurities during the synthesis of linezolid(1).

In a further embodiment, the invention is directed to analytical methodsfor testing the impurity profile of linezolid (1). These methods arealso suitable for analyzing and assaying linezolid (1) and bis-linezolid(4).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the ¹H-NMR spectrum of bis-linezolid (4)

FIG. 2 shows the ¹³C-NMR spectrum of bis-linezolid (4)

FIG. 3 shows the IR spectrum of bis-linezolid (4)

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “reference standard” refers to a compound thatmay be used both for quantitative and qualitative analysis of an activepharmaceutical ingredient. For example, the HPLC retention time of thereference standard compound allows a relative retention time withrespect to the active pharmaceutical ingredient to be determined, thusmaking qualitative analysis possible. Furthermore, the concentration ofthe compound in solution before injection into an HPLC column allows theareas under the HPLC peaks to be compared, thus making quantitativeanalysis possible.

A “reference marker” is used in qualitative analysis to identifycomponents of a mixture based upon their position, e.g., in achromatogram or on a Thin Layer Chromatography (TLC) plate (Strobelpages 921, 922, 953). For this purpose, the compound does notnecessarily have to be added to the mixture if it is present in themixture. A “reference marker” is used only for qualitative analysis,while a reference standard may be used for quantitative or qualitativeanalysis, or both. Hence, a reference marker is a subset of a referencestandard, and is included within the definition of a reference standard.

Prior art methods of preparing linezolid (1) that utilize theintermediate amine (2) have relied on purification steps applied to thelinezolid (1) final product. This was necessary because the reduction ofthe intermediate azide (3) to the intermediate amine (2) in thosemethods did not result in complete conversion of the intermediate azide(3) to the intermediate amine (2) but instead yielded significantamounts of by-products. Those methods yielded undesirably high levels ofcontaminating by-products, e.g., bis-linezolid (4), followingacetylation of the intermediate amine (2) to linezolid (1).

This can be seen in Example 2. Example 2 is a comparative example inwhich the intermediate azide (3) is converted to the intermediate amine(2) by hydrogenation in the solvent ethyl acetate, as in U.S. Pat. No.5,688,792. Following acetylation of the intermediate amine (2), Example2 produced a final product that contained 3.2% bis-linezolid (4).Similarly, when linezolid (1) was prepared directly from theintermediate amine (2) that had been prepared as in Example 4, a highmolecular weight compound was obtained as main impurity (−10%). It wasisolated and identified by MS and NMR as bis-linezolid (4).

The present invention provides isolated bis-linezolid (4).

The bis-linezolid (4) is isolated in at least 98% purity by weight withrespect to other compounds, including linezolid (1). Thus, the isolatedbis-linezolid (4) contains less than about 5%, preferably less thanabout 2%, and even more preferably less than about 1%, by weight oflinezolid (1).

The isolated bis-linezolid (4) of the present invention is characterizedby data selected from: a ¹H NMR spectrum (400 MHz, D₂O+TFA) δ (ppm):2.08(s), 3.63 (m), 3.83 (t), 4.02 (s), 4.14 (m), 4.94 (m), 7.23 (d), 7.58(m); a ¹³C NMR spectrum (400 MHz, CDCl₃) δ (ppm):16.6 (q), 43-50 (s),50.4 (s), 50.9 (s), 68.3 (s), 103.6, 104.0 (d), 111.1 (s) , 118.7 (s),130-140 (s) , 149-153 (d), 171.4 (s); FAB+m/z (MH⁺): 616; and an IRspectrum at 1519, 1572, 1644, 1743, 2825, 2858, 2891, 2965 cm⁻¹.

The isolated bis-linezolid (4) is characterized by ¹H NMR, substantiallyas depicted in FIG. 1. The isolated bis-linezolid (4) is characterizedby ¹³C NMR, substantially as depicted in FIG. 2. The isolatedbis-linezolid (4) is characterized by an IR spectrum, substantially asdepicted in FIG. 3.

The present invention also provides a method for the preparation andisolation of bis-linezolid (4). This method comprises:

-   -   a) combining        R-N-(4-morpholinyl-3-fluorophenyl)-2-oxo-5-oxazolidinyl methyl        azide (3) with an organic solvent and hydrogen gas in the        presence of a catalyst to obtain crude        (S)-N-(4-morpholinyl-3-fluorophenyl)-2-oxo-5-oxazolidinyl-methyl        amine (2);    -   b) combining the crude product of step a) with an organic        solvent to obtain a solution;    -   c) adding acetic anhydride to the solution and maintaining the        reaction mixture for at least 12 hours to obtain crude linezolid        (1);    -   d) combining the crude linezolid (1) of step c) with an organic        solvent to obtain a mixture;    -   e) heating the mixture to reflux; and    -   f) recovering bis-linezolid (4) from the mixture of step e).

Preferably, the organic solvent in steps a), b) and d) is a C₁₋₄ alkylester or an aromatic hydrocarbon. Preferably, the C₁₋₄ alkyl ester isselected from the group consisting of methyl acetate, ethyl acetate,butyl acetate and isobutyl acetate. Preferably, the aromatic hydrocarbonis toluene. Most preferably, the organic solvent in steps a), b) and d)is ethyl acetate. Preferably the catalyst in step a) is Pd/C.

Optionally, an amine, such as pyridine or a tertiary amine, may be addedto the reaction mixture prior to step c). The most preferred amine istriethyl amine.

The isolated bis-linezolid (4) of the present invention is useful as areference standard for linezolid (1). Isolated bis-linezolid (4) may beused to quantify impurities in a linezolid (1) sample. A sample oflinezolid (1) may be spiked with a known amount of purifiedbis-linezolid (4) and analyzed by HPLC to identify peaks associated withthe impurities. Impurity levels can be determined by comparing the areapercent by HPLC of the impurities with the area percent of thebis-linezolid (4) injected in a known amount within linearity ranges. Acontrol sample without added bis-linezolid (4) can be run to determinethe amount of the bis-linezolid (4) peak associated with the addedamount of bis-linezolid (4). Alternatively, at least two samples can beprepared by adding known and differing amounts of bis-linezolid (4) tothe samples. The proportion of the HPLC peak due to the bis-linezolid(4) present in the mixture without the addition of bis-linezolid (4) canbe determined by plotting the HPLC peak area against the amount ofbis-linezolid (4) added to each of the samples, and extrapolating theplot to zero.

The present invention comprises the use of bis-linezolid (4) as areference standard.

The present invention provides a method of determining the amount of animpurity in a sample of linezolid (1) comprising:

-   -   a) measuring by HPLC the area under a peak corresponding to        bis-linezolid (4) in a reference standard comprising a known        amount of bis-linezolid (4);    -   b) measuring by HPLC the area under a peak corresponding to        bis-linezolid (4) in a sample comprising linezolid (1) and        bis-linezolid (4); and    -   c) determining the amount of bis-linezolid (4) in the sample by        comparing the area of step (a) to the area of step (b).

In another embodiment, the present invention provides a method ofdetermining the amount of bis-linezolid (4) in a sample of linezolid (1)comprising:

-   -   a) subjecting a reference standard solution of bis-linezolid (4)        comprising a known amount of bis-linezolid (4) to HPLC for at        least 45 minutes and measuring by HPLC the area under a peak        corresponding to bis-linezolid (4);    -   b) subjecting a sample solution comprising linezolid (1) and        bis-linezolid (4) to HPLC for at least 45 minutes and measuring        by HPLC the area under a peak corresponding to bis-linezolid        (4); and    -   c) determining the amount of bis-linezolid (4) in the sample by        comparing the area of step (a) to the area of step (b).

The present invention discloses that bis-linezolid (4) has an HPLCretention time that is unusually long with respect to linezolid (1).Bis-linezolid (4) is detected at an RRT (relative retention time) ofabout 3.8 relative to linezolid (1).

The present invention also provides a method for detecting bis-linezolid(4) comprising:

-   -   a) providing a preparation of crude linezolid (1) known to        contain or suspected of containing bis-linezolid (4);    -   b) subjecting the preparation of step a) to HPLC;    -   c) determining whether any material has an RRT of about 3.8 with        respect to linezolid (1);        wherein the presence of material with an RRT of about 3.8 with        respect to linezolid (1) indicates that the preparation        contained bis-linezolid (4).

Under certain conditions, step b) includes carrying out the HPLC for atleast about 48 minutes.

The present invention further provides an HPLC method for assayinglinezolid (1) comprising the steps:

-   -   a) combining a linezolid (1) sample with a mixture of        acetonitrile/water diluent having a ratio of about 1:1 to obtain        a solution;    -   b) injecting the solution of step a) into a Hypersil Gold        150×4.6 (or similar) column;    -   c) eluting the sample from the column at about 5 times the        elution time of linezolid (1) using a mixture of about 0.01M        K₂HPO₄: MeOH as an eluent at a gradient of about 80:20 to about        50:50; and    -   d) measuring the bis-linezolid (4) content in the relevant        sample with a UV detector.

Preferably, the bis-linezolid (4) at step d) is measured at a wavelengthof 254 nm. Preferably, the eluting time in step c) is about 45 minutes.

Having described the invention with reference to certain preferredembodiments, other embodiments will become apparent to one skilled inthe art from consideration of the specification. The invention isfurther defined by reference to the following examples describing indetail the preparation of the composition and methods of use of theinvention. It will be apparent to those skilled in the art that manymodifications, both to materials and methods, may be practiced withoutdeparting from the scope of the invention.

EXAMPLES

HPLC method

Column Hypersil Gold 150×4.6, 5 μ

Detection limit: 0.1%

Eluents: 0.01M K₂HPO₄: MeOH A: 80:20 B: 50:50 TABLE 1 Time A B Flow 0100 0 1.5 15 57 43 2 25 35 65 2

Example 1 Isolation of bis-linezolid (4)

Step A: preparation of(S)-N-(4-morpholinyl-3-fluorophenyl)-2-oxo-5-oxazolidinyl-methyl amine(2) enriched with impurities

Into a 1L stainless steel pressure reactor, 10 gR-N-(4-morpholinyl-3-fluorophenyl)-2-oxo-5-oxazolidinyl methyl azide (3)and 455 ml ethyl acetate were charged, followed by 2.5 g of 10% Pd/C.After the addition, the system was blanketed twice with nitrogen.Hydrogen was bubbled into the reaction until a pressure of 1.5 atm. wasreached. The reaction was completed after 6 hrs. The reaction mixturewas filtered. The filtrate was evaporated to dryness to obtain 9.2 g ofcrude (S)-N-(4-morpholinyl-3-fluorophenyl)-2-oxo-5-oxazolidinyl-methylamine (2)

Step B: preparation of crude linezolid (1), enriched with bis-linezolid(4)

Into a 500 ml three-necked reactor equipped, 7.7 g crude(S)-N-(4-morpholinyl-3-fluorophenyl)-2-oxo-5-oxazolidinyl-methyl amine(2) from step A was charged followed by 150 ml ethyl acetate and 7.4 mltriethyl amine. The solution was stirred at RT and then 6.2 ml of aceticanhydride was added. The reaction mixture was stirred overnight and thenfiltered. 5.8 g of dry, crude linezolid (1) was obtained. (yield: 65.8%,bis-linezolid (4) content as per A % in HPLC was 4.1%).

Step C: Isolation of bis-linezolid (4)

Crude linezolid (1) (2.5 g, as prepared in Step B described above), wasmixed with 150 ml ethyl acetate. The mixture was heated to reflux; butit remained turbid. The reaction mixture was filtrated while hot. Thecake contained 0.07 g bis-linezolid (4) (A % (HPLC) 98.1).

Example 2 Comparative Example

In a 1L reactor, 6 gR-N-(4-morpholinyl-3-fluorophenyl)-2-oxo-5-oxazolidinyl-methyl azide (3)was charged with 150 ml ethyl acetate, followed by 0.6 g Pd/C. Thesystem was flushed 3 times with nitrogen and 3 times with hydrogen. Thepressure of hydrogen was set to 1.5 atm. The reaction mixture wasstirred at RT and the reaction followed by TLC or HPLC until completion.The reaction mixture was filtered through celite and the solution wastreated with acetic anhydride in the presence of triethyl amine at RT.The precipitate was filtered and dried to obtain linezolid (1)crystalline Form IV with a 3.2% content of bis-linezolid (4).

Example 3 Use of Bis-Linezolid (4) as a Reference Marker

Two different injections were compared according to the area under thepeak:

a) 10 microliters of a solution containing at least 0.2 mg/ml bislinezolid (1) in acetonitrile : water 1:1 were injected to an HPLCequipped with an Hypersil Gold type column. The retention time of thesample was recorded.

b) 10 microliters of a solution containing at least 0.8 mg/ml linezolid(1) (that might contain impurities such as bis-linezolid (4)) inacetonitrile : water 1:1 were injected to an HPLC equipped with anHypersil Gold type column. The retention time of all sample componentswas recorded.

c) The retention time and the area under the peaks of the impurities inthe linezolid (1) sample were compared. Bis-linezolid (4) eluted atabout 3.8 times longer than linezolid (1).

The relative area percentage of the bis-linezolid (4) peak representsits content in the sample.

1. Isolated bis-linezolid (4).
 2. The isolated bis-linezolid (4) ofclaim 1, characterized by data selected from: ¹H NMR (400 MHz, D₂O+TFA)δ (ppm): 2.08 (s), 3.63 (m), 3.83 (t), 4.02 (s), 4.14 (m), 4.94 (m),7.23 (d), 7.58 (m); ¹³C NMR (400MHz, CDCl₃) δ (ppm):16.6 (q), 43-50 (s),50.4 (s), 50.9 (s), 68.3 (s), 103.6, 104.0 (d), 111.1 (s) , 118.7 (s),130-140 (s), 149-153 (d), 171.4 (s); FAB+m/z (MH⁺): 616; and an IRspectrum at 1519, 1572, 1644, 1743, 2825, 2858, 2891, 2965 cm⁻¹.
 3. Theisolated bis-linezolid (4) of claim 2, characterized by ¹H NMR (400 MHz,D₂O+TFA) δ (ppm): 2.08 (s), 3.63 (m), 3.83 (t), 4.02 (s), 4.14 (m), 4.94(m), 7.23 (d), 7.58 (m).
 4. The isolated bis-linezolid (4) of claim 3,characterized by ¹H NMR substantially as depicted in FIG.
 1. 5. Theisolated bis-linezolid (4) of claim 2, characterized by ¹³C NMR (400MHz, CDCl₃) δ (ppm): 16.6 (q), 43-50 (s), 50.4 (s), 50.9 (s), 68.3 (s),103.6, 104.0 (d), 111.1 (s), 118.7 (s), 130-140 (s), 149-153 (d), 171.4(s).
 6. The isolated bis-linezolid (4) of claim 5, characterized by ¹³CNMR substantially as depicted in FIG.
 2. 7. The isolated bis-linezolid(4) of claim 2, characterized by FAB+m/z (MH⁺):
 616. 8. The isolatedbis-linezolid (4) of claim 2, characterized by an IR spectrum at 1519,1572, 1644, 1743, 2825, 2858, 2891, 2965 cm⁻¹.
 9. The isolatedbis-linezolid (4) of claim 8, characterized by an IR spectrumsubstantially as depicted in FIG.
 3. 10. The isolated bis-linezolid (4)of claim 1, containing less than about 5% by weight of linezolid (1).11. The isolated bis-linezolid (4) of claim 10, containing less thanabout 2% by weight of linezolid (1).
 12. The isolated bis-linezolid (4)of claim 11, containing less than about 1% by weight of linezolid (1).13. A process for preparing the isolated bis-linezolid (4) of claim 1,comprising: a) combiningR-N-(4-morpholinyl-3-fluorophenyl)-2-oxo-5-oxazolidinyl methyl azide (3)with an organic solvent and hydrogen gas in the presence of a catalystto obtain crude(S)-N-(4-morpholinyl-3-fluorophenyl)-2-oxo-5-oxazolidinyl-methyl amine(2); b) combining the crude product of step a) with an organic solventto obtain a solution; c) adding acetic anhydride to the solution andmaintaining the reaction mixture for at least 12 hours to obtain crudelinezolid (1); d) combining the crude linezolid (1) of step c) with anorganic solvent to obtain a mixture; e) heating the mixture to reflux;and f) recovering bis-linezolid (4).
 14. The process of claim 13,wherein the organic solvent in steps a), b) and d) is a C₁₋₄ alkyl esteror an aromatic hydrocarbon.
 15. The process of claim 14, wherein saidorganic solvent is selected from the group consisting of methyl acetate,ethyl acetate, butyl acetate, isobutyl acetate and toluene.
 16. Theprocess of claim 15, wherein said organic solvent is ethyl acetate. 17.The process of claim 13, wherein the catalyst in step a) is Pd/C. 18.The process of claim 13, further comprising adding an amine prior tostep c).
 19. The process of claim 18, wherein said amine is pyridine ora tertiary amine.
 20. The process of claim 19, wherein said amine istriethyl amine.
 21. Use of the isolated bis-linezolid (4) of claim 1 asa reference standard.
 22. A method of determining the amount ofbis-linezolid (4) in a sample of linezolid (1) comprising: a) measuringby HPLC the area under a peak corresponding to bis-linezolid (4) in areference standard comprising a known amount of bis-linezolid (4); b)measuring by HPLC the area under a peak corresponding to bis-linezolid(4) in a sample comprising linezolid (1) and bis-linezolid (4); and c)determining the amount of bis-linezolid (4) in the sample by comparingthe area of step (a) to the area of step (b).
 23. A method ofdetermining the amount of bis-linezolid (4) in a sample of linezolid (1)comprising: a) subjecting a reference standard solution of bis-linezolid(4) comprising a known amount of bis-linezolid (4) to HPLC for at least45 minutes and measuring by HPLC the area under a peak corresponding tobis-linezolid (4); b) subjecting a sample solution comprising linezolid(1) and bis-linezolid (4) to HPLC for at least 45 minutes and measuringby HPLC the area under a peak corresponding to bis-linezolid (4); and c)determining the amount of bis-linezolid (4) in the sample by comparingthe area of step (a) to the area of step (b).
 24. A method for detectingbis-linezolid (4) comprising: a) providing a preparation of crudelinezolid (1) containing bis-linezolid (4); b) subjecting thepreparation of step a) to HPLC; c) determining whether any material hasan RRT of about 3.4 with respect to linezolid (1); wherein the presenceof material with an RRT of about 3.4 with respect to linezolid (1)indicates that the preparation contained bis-linezolid (4).
 25. Theprocess of claim 24, wherein the HPLC in step b) is carried out for atleast about 34 minutes.
 26. An HPLC method for assaying linezolid (1)comprising the steps: a) combining a linezolid (1) sample with a mixtureof acetonitrile/water diluent to obtain a solution; b) injecting thesolution of step a) into a Hypersil Gold 150×4.6 (or similar) column; c)eluting the sample from the column at about 25 min using a mixture ofabout 0.01M K₂HPO₄: MeOH as an eluent at a gradient of about 80:20 toabout 50:50; and d) measuring the bis-linezolid (4) content in therelevant sample with a UV detector.
 27. The process of claim 26, whereinthe bis-linezolid (4) at step d) is measured at a wavelength of 254 nm.